Benzophenoxazine dyes

ABSTRACT

Benzophenoxazine compounds have formula (I) where X is O or NH, Y is NR 1  R 2  or H, R 1  and R 2  are alkyl or -L-A, L is a linker and A may be a reactive group by means of which the compound is linked to a biomolecule. The compounds can be used as fluorescent dyes for labelling biomolecules. ##STR1##

Benzophenoxazine compounds provide a family of fluorescent dyes whichcan be used for labelling biological molecules in various applications.The phenoxazine dyes tend to have large Stokes' shifts of 80-100 nm.This is useful in simplifying the type of filtering needed todifferentiate between excitation and emission wavelengths, and mayenable sensitivity limits to be improved. The benzophenoxazines alsoappear to have particularly good photostability properties.

The benzophenoxazine dye Nile Red(9-diethylaminobenzo[a]phenoxazine-5-one) (FIG. 1) has been shown to beuseful in the fluorescence detection of proteins. Binding of dye toprotein has generally been accomplished through non-covalent,hydrophobic interactions. Fluorescence properties of the dye such asemission wavelength and intensity have been shown to be dependent on thepolarity of the environment and this appears to be the reason for thedifferent spectral properties on binding to different proteins. Its lowfluorescence in water is thought to be due, at least in part, to theformation of dimers and higher aggregates, since there is atime-dependent decrease in intensity on addition to water.

In order to allow specific labelling of proteins and other biomoleculessuch as nucleic acids, attempts have been made to form covalent links,e.g. through the nitrogen at C5 on Nile Blue(5-amino-9-diethylaminobenzo[a]phenoxazine) (FIG. 1). Linkage to NileBlue in this way, however, has been shown to be unsatisfactory becauseof the adverse effect this modification has on the dye's fluorescenceproperties (Mank et al., 1995. Anal. Chem., 67, 1742). Fluorescencequantum yield is reduced by a factor of 10 (from 27 to 2.50) andextinction coefficient is reduced by almost 20 times (from 75000 to4000) (Karnes et al, 1995, Proc. SPIE-Int. Soc. Opt. Eng., 2388, 21).

Bhansali and Kook, Heterocycles, Volume 36, No 6, page 1239, 1993,describe the synthesis of benzophenoxazine compounds substituted at the10 position for therapeutic applications by intercalating into DNA. Itis also stated in U.S. Pat. No. 5,283,330 that the alanyl substitutedcompound can act as a protein dye. In neither of these cases was thisclass of compound covalently linked to the DNA or protein target, norare the fluorescence properties of the compounds mentioned or described.

The benzophenoxazine ring numbering scheme is also shown in FIG. 1. Thisinvention is concerned with novel derivatives of benzophenoxazines whichhave substituents at the 2 and/or 10 and/or the 9 position, whichsubstituents do not cause a significant loss of fluorescence. Reactivegroups at all three positions can be used for linkage to tracermolecules such as biomolecules or to introduce water solubilisingfunctions in order to improve compatibility with aqueous systems or tochange the fluorescence emission wavelength of the compound.

According to this invention there is provided a benzophenoxazinecompound having the formula ##STR2## where X is O or NH or N-alkyl orN-aryl or N-alkenyl,

Y is NR¹ R² or H,

R¹ and R² are the same or different and each is C1-C12 aryl, alkenyl oralkyl or is -L-A,

in at least one -L-A:

each L is a linker chain of 0-20 carbon atoms and which may contain oneor more O or N or S atoms, and

each A may be amine or amide or --CN or alcohol or thiol or carboxyl orsulfonate or phosphate, or a reactive group by means of which thebenzophenoxazine compound may be covalently linked to a biomolecule or agroup which enhances or reduces water solubility or provides electrondonor or withdrawal properties to modify the spectral characteristics ofthe dye,

and each other -L-A is H or C1-C20 alkyl.

In these compounds, the 5-substituent X may be O as in Nile Red or NH asin Nile Blue, or may be N-alkyl, N-aryl or N-alkenyl as in related dyessuch as oxazine 170.

At the 9-position, each N-substituent R¹ and R² is the same or differentC1-C12 alkyl or aryl or alkenyl or is -L-A.

In at least one -L-A group, L is a linker chain of 0 to 20 carbon atomsand which may include O or N or S atoms. Preferably the chain is atleast three atoms in length, so as to avoid possible steric inhibitionof reactions, e.g. --OC₅ H₁₀ -- and --OCOC₄ H₈ -- and --C₂ H₄ CO--.

The compound contains at least one group A which is an amine or amide or--CN or alcohol or thiol or carboxyl or sulfonate or phosphate or areactive group by means of which the benzophenoxazine compound may becovalently linked to a biomolecule, or a group which enhances (e.g.polyether) or reduces (e.g. alkyl) water solubility or provides electrondonor or withdrawal properties to modify the spectral characteristics ofthe dye, e.g. halogen, alkoxy, nitro or cyano. See The Chemistry ofSynthetic Dyes, Venkataraman, Academic Press, N.Y. 1971, 4, Chapter 5part iiic, p 228-240, particularly Table 1 on page 230. Preferredbiomolecules are nucleosides, nucleotides and analogues thereof,oligonucleotides and nucleic acids, and also peptides, proteins,polysaccharides, lipids, sugars and other small molecules.

In another aspect, the invention provides a complex of abenzophenoxazine compound and a biomolecule, the benzophenoxazinecompound having the formula ##STR3## where X, Y, R¹, R², L and A are asdefined above, and wherein the benzophenoxazine compound is covalentlylinked to the biomolecule through a reactive group A. These linkages canbe conventional. For example, a carboxylate group A may be converted toa succinimide ester for reaction with an amine group of a nucleoside ornucleotide or peptide. Examples of other reactive groups most commonlyused are isothiocyanate, iodoacetamides, sulphonyl chlorides,maleimides, phosphoramidites, phosphonates and azides. Preferably thelinkage is through the 2-position, or through the 9-position or the10-position of the benzophenoxazine molecule.

Any other group -L-A may optionally be hydrogen or C1-C20 alkyl.

EXPERIMENTAL

Commercially available solvents and reagents were used throughoutwithout further purification, except for those detailed below which werepurified as described. DMF was dried by stirring over calcium hydridefor 15 h, decanted and distilled under reduced pressure before storageover molecular sieves under nitrogen. Methanol was distilled frommagnesium turnings and iodine.

Analytical thin layer chromatography was carried out usingaluminium-backed plates coated with Merck Kieselgel 60 GF₂₅₄. Plateswere visualised under UV light (at 254 and/or 360 nm). Flashchromatography was carried out using Merck Kieselgel 60 H silica orMatrex silica 60. Pressure was applied at the column head with handbellows. Samples were applied pre-adsorbed on silica. HPLC purificationwas carried out using either a C18 reverse phase column equilibrated inwater with methanol elution or a PRP1 column equilibrated with 2%ammonia and using methanol elution.

IR spectra were recorded in the range 4000-600 cm⁻¹ using a NicoletFT-205 spectrometer, with internal calibration. Spectra were recorded asKBr discs. UV/visible spectra were obtained using a Shimadzu UV-160spectrophotometer. Uncorrected fluorescence spectra were recorded asdilute solutions on a Perkin Elmer LS50B luminescence spectrometer or aHitachi F4500 using optiglass fluorescence cells. ¹ H and ¹³ C NMRspectra were recorded on HPLC purified samples using Bruker AC-250,Bruker DPX400 and Bruker WH400 instruments (SERC NMR SpectroscopyCentre, Warwick, UK) and a Jeol GSX-270 instrument; J values wererecorded in Hz. High and low-resolution mass spectra were recorded on aKratos MS80 instrument or on a VG Analytical ZAB-E instrument (SERC MassSpectrometry Service, Swansea). Melting points were measured on anElectrothermal digital melting point apparatus or on a Reichert-Koflerhot stage apparatus and are uncorrected.

EXAMPLE 1 Introduction of Functional Groups at the 2 Position9-Diethylamino-2-hydroxy-5H-benzo[a]phenoxazin-5-one (I)

5-Diethylamino-2-nitrosophenol hydrochloride (1.14 g, 4.96+mmol) and1,6-dihydroxynaphthalene (0.19 g, 4.94 mmol) were refluxed in DMF (100ml) for 4 h. The DMF was removed under reduced pressure. The crudemixture was purified by flash chromatography (ethyl acetate--1:1 ethylacetate:isopropanol elution), to yield a dark green crystalline solid.

Yield (1.1 g, 65%); m.p. >300° C.; (Found: M⁺ 334.1317. C₂₀ H₁₈ N₂ O₃requires 334.1317); ν_(max) (KBr)/cm⁻¹ 3375, 2964, 1645, 1590, 1561;λ_(max) (MeOH)/nm 547 (ε30366), 326 (5762), 265 (27555), 210 (23380);δ_(H) (400 MHz; DMSO d₆) 10.42 (1H, broad s, OH), 7.96 (1H, d, J=8.6 Hz,4-H), 7.87 (1H, d, J=2.4 Hz, 1-H), 7.55 (1H, d, J=9.0 Hz, 11-H), 7.08(1H, dd, J=8.6 Hz, J=2.5 Hz. 3-H), 6.77 (1H, dd, J=9.0 Hz, J=2.5 Hz,10-H), 6.61 (1H, d, J=2.5 Hz, 8-H), 6.13 (1H, s, 6-H), 3.48 (4H, q,J=7.0 Hz, N(CH₂ CH₃)₂), 1.15 (6H, t, J=7.0 Hz, N(CH₂ CH₃)₂); δ_(C)(100.6 MHz; DMSO d₆) 181.64 (CO), 160.69, 151.66, 150.74, 146.47,138.77, 133.82, 130.88 (CH), 127.52 (CH), 123.92, 118.42 (CH), 109.96(CH), 108.18 (CH), 104.15 (CH), 96.09 (CH), 44.48 (CH₂ CH₃), 12.52 (CH₂CH3); m/z (EI) 335 (MH⁺, 20%), 334 (M⁺, 63), 320 (25), 319 (100), 291(26); fluorescence in methanol strong.

9-Diethylamino-5-oxo-5H-benzo[a]phenoxazine-2-carboxylic Acid (II)

This was made similarly to the hydroxy derivative above except that thestarting materials were 5-diethylamino-2-nitrosophenol hydrochloride and1-hydroxynaphthalene-6-carboxylic acid.

EXAMPLE 2 Derivatisation of 2-Hydroxyl Analogue with a 6 Carbon Linkerthrough Ester Formation9-Diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yl(5-carboxy)pentanoate(III)

A mixture of the 9-diethylamino-2-hydroxy-5H-benzo[a]phenoxazin-5-one(0.096 g, 0.28 mmol), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-p-toluene sulfonate (0.18 g, 0.42 mmol), adipic acid (0.046 g,0.31 mmol) and a catalytic quantity of 4-dimethylaminopyridine werestirred overnight in dichloromethane (30 ml). The crude mixture waswashed with water, dried (Na₂ SO₄) and concentrated. The residue waspurified by column chromatography (ethyl acetate elution) to yield theacid as a dark red solid.

Yield (0.08 g, 60%); m.p. 85-87° C.; ν_(max) (film)/cm⁻¹ 3347, 3055,2985, 2930, 1757, 1639, 1586; λ_(max) (MeOH)/nm 558 (ε22222),267(28090), 208 (17094); δ_(H) (250 MHz; CDCl₃) 8.32 (2H, 4-H, 1-H), 7.56(1H, d, J=9.1 Hz, 11-H), 7.34 (1H, dd, J=8.7 Hz, J=2.3 Hz, 3-H), 6.62(1H, dd, J=9.2 Hz, J=2.7 Hz, 10-H), 6.44 (1H, d, J=2.7 Hz, 8-H), 6.40(1H, s, 6-H), 3.47 (4H, q, J=7.1 Hz, N(CH₂ CH₃)₂), 2.68 (2H, t, J=7.0Hz, COCH₂), 2.49 (2H, t, J=6.9 Hz, COCH₂), 1.26 (6H, t, J=7.1 Hz, N(CH₂CH₃)₂); δ_(H) (100.6 MHZ; CDCl₃) 182.91 (CO), 171.41 (CO), 153.18,152.41, 151.05, 146.94, 139.05, 133.72, 131.30 (CH), 129.46, 127.66(CH), 124.99, 123.48 (CH), 116.36 (CH), 109.93 (CH), 105.50 (CH), 96.39(CH), 45.14 (CH₂ CH₃), 34.05 (CH₂), 29.70 (CH₂), 24.24 (CH₂), 24.14(CH₂), 12.62 (CH₂ CH₃); m/z (Cl) 463 (MH⁺, 84%), 335 (100), 146 (13), 74(18) fluorescence in methanol strong.

EXAMPLE 3 Derivatisation of 2-Hydroxyl Analogue with a 6 Carbon Linkerthrough Ether Formation Benzyl6-(9-diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoate (IV)

A mixture of 9-diethylamino-2-hydroxy-5H-benzo[a]phenoxazin-5-one (0.1g, 0.3 mmol), potassium carbonate (0.12 g, 0.9 mmol) and benzyl6-bromohexanoate (0.11 g, 0.38 mmol) in DMF (50 ml) was refluxed for 4h. Residual potassium carbonate was filtered off and the filtrateconcentrated under reduced pressure. The crude residue was purified bycolumn chromatography (petrol -petrol: ether, 1:1, 10% gradient elution)to yield a green crystalline solid.

m.p. 146-148° C.; (Found: M⁺ 538.2470. C₃₃ H₃₄ N₂ O₅ requires 538.2467);ν_(max) (KBr)/cm⁻¹ 2937, 2865, 1734, 1595; λ_(max) (MeOH)/nm 551(ε27169), 267 (28144), 210 (30531); δ_(H) (250 MHz; CDCl₃) 8.20 (1H, d,J=8.7 Hz, 4-H), 8.02 (1H, d, J=2.5 Hz, 1-H), 7.59 (1H, d, J=9.1 Hz,11-H), 7.35 (5H, broad s, Ph), 7.13 (1H, dd, J=8.7 Hz, J=2.6 Hz, 3-H),6.63 (1H, dd, J=9.1 Hz, J=2.6 Hz, 10-H), 6.44 (1H, d, J=2.6 Hz, 8-H),6.28 (1H, s, 6-H), 5.13 (2H, s, CH₂ Ph), 4.14 (2H, t, J=6.2 Hz, ArOCH₂),3.45 (4H, q, J=7.0 Hz, N(CH₂ CH₃)₂), 2.43 (2H, t, J=7.3 Hz, CH₂ CO₂ Bn),1.85 (2H, m), 1.76 (2H, m), 1.58 (2H, m), 1.25 (6H, t, J=7.0 Hz, N(CH₂CH₃)₂); δ_(C) (100.6 MHz; CDCl₃)184.14 (CO). 1173.34 (CO2Bn), 161.60,151.90, 150.59, 146.67, 139.76, 133.95, 130.95 (CH), 128.50 (CH), 128.14(CH), 127.56 (CH), 125.44, 124.57, 118.13 (CH), 109.40 (CH), 106.39(CH), 105.10 (CH), 96.12 (CH), 67.93 (CH₂), 66.09 (CH₂), 44.97 (N(CH₂CH₃)₂), 34.14 (CH₂), 28.82 (CH₂), 25.59 (CH₂), 24.64 (CH₂), 12.57 (N(CH₂CH₃)₂); m/z (Cl) 539 (MH⁺, 46%), 335 (22), 165 (22), 132 (100);fluorescence in methanol strong.

Methyl 6-(9-Diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoate(V)

This was formed by transesterification from the benzyl ester using thesame procedure described for formation of the benzyl ester (IV) butusing methanol as solvent.

m.p. 152-154° C.; ν_(max) (KBr)/cm⁻¹ 2929, 2868, 1739, 1620, 1601, 1581;λ_(max) (MeOH)/nm 553 (ε27800), 265 (27433), 212 (21459); δ_(H) (400MHz; CDCl₃) 8.19 (1H, d, J=8.7 Hz, 4-H), 8.01 (1H, d, J=2.6 Hz, 1-H),7.57 (1H, d, J=9.0 Hz, 11-H), 7.13 (1H, dd, J=8.7 Hz, J=2.6 Hz, 3-H),6.62 (1H, dd, J=9.1 Hz, J=2.7 Hz, 10-H), 6.42 (1H, d, J=2.7 Hz, H8),6.26 (1H, s, 6-H), 4.16 (2H, t, J=6.4 Hz, ArOCH₂), 3.68 (3H, s, CO₂ Me),3.44 (4H, q, J=7.1 Hz, N(CH₂ CH₃)₂), 2.38 (2H, t, J=7.4 Hz, CH₂ CO₂ Me),1.88 (2H, m), 1.74 (2H, m), 1.58 (2H, m), 1.25 (6H, t, J=7.1 Hz, ν (CH₂CH₃)₂); δ_(C) (100.6 MHz; CDCl₃) 183.51 (CO), 174.30 (CO), 152.30,150.98, 147.09, 140.33, 134.33, 131.321, 128.83 (CH), 125.88 (CH),124.95, 122.15, 117.61 (CH), 109.74 (CH), 106.88 (CH), 105.56 (CH),96.58 (CH), 68.38 (CH₂), 51.78 (CO₂ Me), 44.98 (CH₂), 34.26 (CH₂), 29.18(CH₂), 25.97 (CH₂), 24.98 (NCH₂ CH3), 12.90 (NCH₂ CH₃); m/z (ES) 463(MH⁺, 28%), 234 (14), 194 (28), 179 (37), 98 (67), 84 (100);fluorescence in methanol strong.

Ethyl 6-(9-Diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoate(VI)

A mixture of 9-diethylamino-2-hydroxy-5H-benzo[a]phenoxazin-5-one (0.27g, 0.81 mmol), potassium carbonate (0.34 g, 2.46 mmol) and ethyl6-bromohexanoate (0.28 g, 1.26 mmol) in methanol (100 ml) was refluxedfor 2 days. After this time full conversion was observed by TLC. Excesspotassium carbonate was filtered off and the filtrate concentrated. Thecrude residue was evaporated and purified by column chromatography(ether elution), to yield a red crystalline solid.

Yield (0.21 g, 53%); m.p. 158-160° C.; (Found: M⁺ 476.2311. C₂₈ H₃₂ N₂O₅ requires 476.2311); ν_(max) (KBr)/cm⁻¹ 2927, 1735, 1628, 1600, 1582;λ_(max) (MeOH)/nm 552 (ε44064), 267 (40664), 208 (38216); δ_(H) (250MHz; CDCl₃) 8.21 (1H, d, J=8.7 Hz, 4-H), 8.04 (1H, d, J=2.6 Hz, 1-H),7.60 (1H, d, J=9.1 Hz, 11-H), 7.15 (1H, dd, J=8.7 Hz, J=2.5 Hz, 3-H),6.65 (1H, dd, J=9.1 Hz, J=2.7 Hz, 10-H). 6.45 (1H, d, J=2.7 Hz, 8-H),6.29 (1H, s, 6-H), 4.15 (4H, m, CO₂ CH₂ CH₃, ArOCH₂), 3.47 (4H, q, J=7.1Hz, N(CH₂ CH₃)₂), 2.36 (2H, t, J=7.2 Hz, CH₂ CO₂ Et), 1.89 (2H, m), 1.75(2H, m), 1.57 (2H, m), 1.26 (9H, t, J=7.1 Hz, N(CH₂ CH₃)₂, CO₂ CH₂ CH₃);δ_(C) (62.9 MHz; CDCl₃) 183.14 (CO), 173.86 (CO₂ Et), 161.63, 151.92,150.60, 146.69, 140.00, 133.95, 130.94 (CH), 127.57 (CH), 125.46,124.57, 118.14, 109.39 (CH), 106.42 (CH), 105.13 (CH), 96.15 (CH), 67.95(CH₂), 60.20 (CH₂), 44.97 (CH₂), 34.18 (CH₂), 28.84 (CH₂), 25.61 (CH₂),24.65 (CH₂), 14.19 (CH₃), 12.55 (CH₃); m/z (EI) 477 (MH⁺, 15%), 476 (M⁺,67), 461 (73), 431 (13), 291 (15), 233 (17); fluorescence in methanolstrong.

6-(9-Diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoic Acid (VI)

The ethyl ester (ethyl6-(9-diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoate) (0.055g, 0.11 mmol) was suspended in ammonium dihydrogen phosphate (0.1 M, 5ml, pH 7) and porcine liver esterase (0.6 ml, 1500 units) added. Themixture was stirred at 37° C. for 7 days and checked by TLC. The waterwas removed under reduced pressure. The residue was dissolved inmethanol and the enzyme extract filtered off through celite. The crudemixture was purified by reverse phase chromatography (methanol elution),to yield a dark red crystalline solid.

Yield (0.032 g, 62%); m.p. 167-169° C.; (Found: MH⁺ 449.2076. C₂₆ H₂₈ N₂O₅ +H requires 449.2076); ν_(max) (KBr)/cm⁻¹ 3435, 2953, 1653, 1638,1619, 1593; λ_(max) (MeOH)/nm 550 (ε30423), 325 (5824), 267 (28753), 210(20759); δ_(H) (250 MHz; MeOH d₄) 8.02 (1H, d, J=8.8 Hz, 4-H), 7.92 (1H,d, J=2.4 Hz, 1-H), 7.52 (1H, d, J=9.2 Hz, 11-H), 7.10 (1H, dd, J=8.8 Hz,J=2.5 Hz, 3-H), 6.76 (1H, dd, J=9.2 Hz, J=2.5 Hz, 10-H), 6.51 (1H, d,J=2.5 Hz, 8-H), 6.14 (1H, s, 6-H), 4.09 (2H, t, J=7.7 Hz, ArOCH₂), 3.49(4H, q, J=7.1 Hz, N(CH₂ CH₃)₂), 2.33 (2H, t, J=7.1 Hz, CH₂ CO₂ H), 1.86(2H, m), 1.69 (2H, m), 1.56 (2H, m), 1.24 (6H, t, J=7.1 Hz, N(CH₂CH₃)₂); δ_(C) (100.6 MHz; DMSO d₆) 181.49 (CO), 174.77 (CO₂ H), 161.34,151.78, 150.88, 146.54, 138.33, 133.64, 131.05 (CH), 127.30 (CH),124.91, 124.00, 117.98 (CH), 110.12 (CH), 106.25 (CH), 104.14 (CH),96.04 (CH), 67.95 (CH₂), 44.54 (CH₂), 34.01 (CH₂), 28.50 (CH₂), 25.26(CH₂), 24.51 (CH₂), 12.57 (CH₃); m/z (FAB) 449 (MH⁺, 54%), 330 (42), 308(73), 290 (56), 165 (65); fluorescence in methanol strong.

EXAMPLE 4 Labelling of Model Biological Molecules Synthesis of theActive Ester (Succinyl6-(9-Diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoate) (VIII)

6-(9-Diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoic acid(0.12g, 0.26 mmol), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-p-toluenesulfonate (0.34 g, 0.8 mmol), N-hydroxysuccinimide (0.09g, 0.8 mmol) and a catalytic quantity of 4-dimethylaminopyridine werestirred overnight in dichloromethane (25 ml). The solvent was removedunder reduced pressure and the residue purified by column chromatography(ether) elution) to yield the active ester as a dark red solid.

Yield (0.13, 92%); m.p. 166-168° C.; (Found: M⁺ 545.2160. C₃₀ H₃₁ N₃ O₇requires 545.2162); ν_(max) (KBr)/cm⁻¹ 3400, 1737, 1623, 1617, 1589;λ_(max) (MeOH/nm) 552 (ε30929), 325 (5359), 268 (27886), 210 (34608);δ_(H) (250 MHz; CDCl₃) 8.20 (1H, d, J=8.7 Hz, 4-H), 8.02 (1H, d, J=2.5Hz, 1-H) 7.58 (1H, d, J=9.2 Hz, 11-H), 7.15 (1H, dd, J=8.7 Hz, J=2.5 Hz,3-H), 6.63 (1H, dd, J=9.2 Hz, J=2.7 Hz, 10-H), 6.43 (1H, d, J=2.7 Hz,8-H), 6.28 (1H, s,6-H), 4.18 (2H, t, J=6.2 Hz, ArOCH₂), 3.45 (4H, q,J=7.1 Hz, N(CH₂ CH₃)₂), 2.84 (4H, s, COCH₂ CH₂ CO), 2.68 (2H, t, J=7.2Hz, CH₂ CO₂ N(COCH₂)₂),1.88 (4H, m), 1.68 (2H, m),1.25 (6H, t, J=7.1 Hz,N(CH₂ CH₃)₂); δ_(C) (100.6 MHz; CDCl₃) 169.46 (CO), 168.85 (CO), 162.09(CO), 152.42, 151.10, 147.22, 140.50, 134.45 (CH), 131.44 (CH), 128.11,126.03, 125.09, 118.68 (CH), 109.86 (CH), 107.02 (CH), 105.68 (CH),96.72 (CH), 68.28 (CH₂), 45.42 (CH₂), 31.30 (CH₂), 29.12 (CH₂), 25.98(CH₂),25.77 (CH₂), 24.78 (CH₂), 13.00 (N(CH₂ CH₃)₂); m/z (EI) 546 (MH⁺,26%), 545 (M⁺, 85), 530 (40), 430 (62), 415 (88), 262 (52), 233 (100);fluorescence in methanol strong.

Reaction of Active Ester with Allylamine (IX)

(a) Homogeneous reaction

To a stirred solution of the active ester VIII (0.039 g, 0.07 mmol) anda catalytic quantity of 4-dimethylaminopyridine in dichloromethane wasadded dropwise allylamine (0.026 ml, 0.35 mmol).

The mixture was stirred overnight. The solvent was removed under reducedpressure and the residue purified by column chromatography (ethylacetate elution) to yield the allyl amide as a dark red solid (0.02 g,59%).

(b) Biphasic Reaction

To a stirred solution of the active ester (0.06 g, 0.11 mmol) indichloromethane (15 ml) was added a solution of sodium hydrogencarbonate (0.1 m, 8 ml) and sodium carbonate (0.1M, 8 ml) containingallyl amine (0.04 ml, 0.54 mmol) and tetrabutylammonium hydrogen sulfate(0.037 g, 0.11 mmol). The mixture was stirred for 2 h and followed byTLC. The layers were separated and the dichloromethane layer was dried(MgSO₄) and concentrated to yield the allylamide as a dark red solid(0.37 g, 69%).

6-(9-Diethylamino-5oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoic AcidTryptophan Methyl Ester Amide (X)

A solution of the active ester VIII (0.035 g, 0.06 mmol), L-tryptophanmethyl ester hydrochloride (0.049 g, 0.19 mmol) and pyridine (0.015 g,0.19 mmol) in dichloromethane (20 ml) was stirred overnight. The solventwas removed under reduced pressure and the residue purified by columnchromatography (ethyl acetate elution) to yield the trytophan amide as adark red crystalline solid.

Yield (0.027 g, 65%); m.p. 75-77° C.; ν_(max) (KBr)/cm⁻¹ 3421, 3300,2930, 1750, 1735, 1654, 1617, 1594, 1572; ν_(max) (MeOH)/nm 551(ε38033), 326 (7308), 268 (42555), 220 (70697); δ_(H) (250 MHz; CDCl₃)8.54 (1H, broad s, NH), 8.18 (1H, d, J=8.7 Hz, 4-H), 8.00 (1H, J=2.5 Hz,1-H), 7.58 (1H, d, J=9.1 Hz, 11-H), 7.56 (1H, m), 7.36 (1H, m), 7.16(4H, m), 6.61 (1H, dd, J=9.1 Hz, J=2.7 Hz, 10-H), 6.43 (1H, d, J=2.6 Hz,8-H), 6.28 (1H, s, 6-H), 6.05 (1H, d, J=7.8 Hz, CONH), 4.97 (1H, td,J=7.8 Hz, J=5.6 Hz, HNCHCO₂ Me), 4.04 (2H, t, J=6.4 Hz, ArCH₂), 3.71(3H, s, OMe), 3.45 (4H, q, J=7.1 Hz, N(CH₂ CH₃)₂), 3.32 (2H, t, J=5.6Hz, CH(CO₂ Me)CH₂), 2.22 (2H, t, J=7.4 Hz, CH₂ CON), 1.81.(2H, m), 1.72(2H, m), 1.63 (2H, m), 1.25 (6H, t, J=7.1 z, N(CH₂ CH₃)₂); δ_(C) (100.6MHz; CDCl₃) 183.67 (CO), 172.93 (CONH), 162.15 (CO₂ Me), 152.46, 151.16,147.22, 140.35, 136.59, 134.41, 131.38 (CH), 128.12 (CH), 125.92,125.92, 125.10, 123.18 (CH), 122.60 (CH), 120.01 (CH), 119.88, 119.12,118.86 (CH), 118.70 (CH), 111.79 (CH), 110.40, 109.97 (CH), 106.94 (CH),105.61 (CH), 96.70 (CH), 68.42 (CH₂), 53.28 (CHCO₂ CH₃), 52.70 (CO₂ Me),45.43 (NCH₂ CH₃), 36.78 (CH₂), 29.23 (CH₂), 28.00 (CH₂), 25.92 (CH₂),25.52 (CH₂), 13.00 (N(CH₂ CH₃)₂); m/z (ES) 649 (MH⁺, 68%), 489 (9), 348(23), 263 (100); fluorescence in methanol strong.

6-(9-Diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoic AcidGlycine Tert-Butyl Ester Amide (XI)

A mixture of6-(9-diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoic acid (10mg, 22 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide(ETC) (14 mg, 45 mmol) and N-hydroxysulfosuccinimide sodium salt (15 mg,67 mmol) in DMF (1 ml) was stirred at room temperature. After 2.5 h afurther portion of ETC (50 mg, 168 mmol) was added. The mixture wasstirred at room temperature overnight and a solution of tert-butylglycine hydrochloride (19 mg, 112 mmol) in pH 9.2 buffer (NaHCO₃ /Na₂CO₃) (1.5 ml) was added dropwise. After 3 h the mixture was concentratedin vacuo and purified by flash column chromatography (C18, 50%methanol/water to 80% methanol/water gradient) to afford the titlecompound as a dark red solid (11 mg, 88%).

d_(H) (270 MHz; CDCl₃) 8.21 (1H, d, J=8.8 Hz, 4-H), 8.03 (1H, d, J=2.5Hz, 1-H), 7.61 (1H, d, J=9.1 Hz, 11-H), 7.15 (1H, dd, J=8.6 Hz and 2.6Hz, 3-H), 6.65 (1H, dd, J=9.3 Hz and 2.7 Hz, 10-H), 6.45 (1H, d, J=2.8Hz, 8-H), 6.29 (1H, s, 6-H ) 6.00 (1H, br t, J=5.2 Hz, NH), 4.17 (2H, t,J=6.3 Hz, ArOCH₂), 3.95 (2H, d, J=5.2 Hz, NHCH₂), 3.47 (4H, q, J=7.1 Hz,N(CH₂ CH₃)₂), 2.30 (2H, d, J=7.4 Hz, CH₂ CON), 1.95-1.68 (4H, m),1.68-1.50 (2H, m), 1.47 (9H, s, Bu^(t)) and 1.26 (2H, t, J=7 Hz, N(CH₂CH₃)₂) fluorescence in methanol strong.

EXAMPLE 5 Derivatisation at the 10 Position with Alkylcarboxy orAlkylamino Groups 10-(2-Aminoethyl)-5H-benzo[a]phenoxazin-5-one (XII)

This was synthesised according to the methods of Bhansali and Kook(Petrocycles, 36, 1239-51, 1993).

10-(2-Carboxyethyl)-5H-benzo[a]phenoxazin-5-one (XIII)

This was synthesised according to the methods of Bhansali and Kook(1993) but using 3-(4-hydroxyphenyl)propionic acid as the phenolicsubstrate.

EXAMPLE 6 Introduction of Water-Solubilising Functionality into theDialkylamino Grouping at the 9 Position9(N-Ethyl-(N-3-sulfonylpropyl))amino-2-hydroxy-5H-benzo[a]phenoxazin-5-one(XIV)

3-(N-Ethyl-3-hydroxy4-nitrosoanilino)-propanesulfonic acid hydrochloride(0.53 g, 1.6 mmol) and 1,6-dihydroxynaphthalene (0.26 g, 1.6 mmol) wererefluxed in DMF (40 ml) for 4 h. The DMF was removed under reducedpressure. The crude mixture was purified by flash chromatography (ethylacetate-1:1 ethyl acetate: methanol, 10% gradient elution) to yield adark green crystalline solid.

Yield (0.54 g, 79%); m.p. 147-149° C.; ν_(max) (KBr)/cm⁻¹ 3421, 2970,1639, 1617, 1592, 1560; λ_(max) (MeOH)/nm 547 (ε28969), 326 (5541), 264(26761), 212 (21805); δ_(H) (400 MHz; MeOH d₄) 7.92 (1H, d, J=8.6 Hz,4-H), 7.76 (1H, d, J=2.4 Hz, 1-H), 7.35 (1H, d, J=9.1 Hz, 11-H), 6.98(1H, dd, J=8.6 Hz, J=2.5 Hz, 3-H), 6.68 (1H, dd, J=9.1 Hz, J=2.5 Hz,10-H), 6.41 (1H, d, J=2.5 Hz, 8-H), 6.03 (1H, s, 6-H), 3.53 (2H, t,J=7.7 Hz, HO₃ SCH₂ CH₂ CH₂ N), 3.46 (2H, q, J=7.0 Hz, CH₃ CH₂ N), 2.91(2H, t, J=7.2 Hz, HO₃ SCH₂), 2.11 (2H, m, HO₃ SCH₂ CH₂ CH₂ N), 1.22 (3H,t, J=7.0 Hz, CH₃ CH₂ N); δ_(C) (100 MHz; MeOH d₄) 182.91 (CO), 160.09,151.65, 150.80, 145.82, 137.56, 133.52, 130.15 (CH), 126.49 (CH),124.24, 123.14, 117.02 (CH), 109.73 (CH), 107.60 (CH), 102.64 (CH),95.28 (CH), 48.47 (CH₂), 47.67 (CH₂), 44.40 (CH₂), 22.21 (CH₂), 10.69(CH₃); m/z (ES) 428 (M⁻, 23%), 427 (M-H⁻, 100); fluorescence in methanolstrong.

Ethyl6-(9(N-ethyl-(N-3-sulfonylpropyl))amino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoate(XV)

A mixture of ⁹(N-ethyl-(N-3-sulfonylpropyl))amino-2-hydroxy-5H-benzo[a]phenoxazin-5-one(0.34 g, 0.86 mmol), potassium carbonate (0.56 g, 4.06 mmol) and ethyl6-bromohexanoate (0.95 g, 4.26 mmol) in DMF (25 ml) was refluxedovernight. Excess potassium carbonate was filtered off and the filtrateconcentrated. The crude residue was purified by column chromatography(ethyl acetate--1:1 ethyl acetate:methanol, 10% gradient elution) toyield a red crystalline solid.

Yield (0.4 g, 87%); m.p. 150-152° C.; ν_(max) (KBr)/cm⁻¹ 3442, 2928,2869, 1733, 1619, 1596; λ_(max) (MeOH)/nm 552 (E 20732), 326 (3963), 266(19539), 212 (14886); δ_(H) (400 MHz; MeOH d₄) 7.93 (1H, d, J=8.6 Hz,4-H), 7.81 (1H, d, J=2.5 Hz, 1-H), 7.44 (1H, d, J=9.1 Hz, 11-H), 7.00(1H, dd, J=8.7 Hz, J=2.5 Hz, 3-H), 6.76 (1H, dd, J=9.3 Hz, J=2.5 Hz,10-H), 6.50 (1H,, d, J=2.5 Hz, 8-H), 6.05 (1H, s, 6-H), 4.04 (4H, m,ArOCH₂, CO₂ CH₂ CH₃), 3.50 (4H, m, HO₃ SCH₂ CH₂ CH₂ N, NCH₂ CH₃), 2.82(2H, t, J=7.2 Hz, HO₃ SCH₂ CH₂ CH₂ N), 2.29 (2H, t, J=7.3 Hz, CH₂ CO₂Et), 2.03 (2H, m, HO₃ SCH₂ CH₂ CH₂ N), 1.77 (2H, m), 1.62 (2H, m), 1.47(2H, m), 1.16 (6H, m, NCH₂ CH₃, CO₂ CH₂ CH₃); δ_(C) (100.6 MHz; MeOH d₄)184.18 (CO), 174.70 (CO₂ Et), 162.54, 153.12, 152.29, 147.35, 138.87,134.64, 131.58 (CH), 127.60 (CH), 125.55, 125.35, 118.41 (CH), 111.20(CH), 106.65 (CH), 96.67 (CH). 68.50 (CH₂), 60.66 (CH₂), 49.26 (CH₂),45.72 (CH₂), 34.26 (CH₂), 29.14 (CH₂), 25.03 (CH₂), 23.44 (CH₂), 13.78(CH₃), 11.94 (CH₃); m/z (ES) 570 (M⁻, 39%), 569 (M-H⁻, 100);fluorescence in methanol strong.

Benzyl6-(9-(N-ethyl-(N-3-sulfonylpropyl))amino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoate(XVI)

A mixture of9(N-ethyl-(N-3-sulfonylpropyl))amino-2-hydroxy-5H-benzo[a]phenoxazin-5-one(0.54 g, 1.26 mmol), potassium carbonate (0.52 g, 3.76 mmol) and benzyl6-bromohexanoate (0.47 g, 1.65 mmol) in DMF (25 ml) was refluxedovernight. Excess potassium carbonate was filtered off and the filtrateconcentrated.. The crude residue was purified by column chromatography(ethyl acetate--1:1 ethyl acetate:methanol, 10% gradient elution) toyield a red crystalline solid.

Yield (0.4 g, 51%); m.p. 156-158° C.; ν_(max) (KBr)/cm⁻¹ 3448, 2938,1735, 1640, 1619, 1595; λ_(max) (MeOH)/nm 551 (ε7973), 326 (1546), 267(7535), 209 (7496); δ_(H) (400 MHz; MeOH d₄) 7.98 (1H, d, J=8.7 Hz,4-H), 7.83 20 (1H, d, J=2.4 Hz, 1-H), 7.48 (1H, d, J=9.1 Hz, 11-H), 7.30(5H, m, Ph), 7.03 (1H, dd, J=8.8 Hz, J=2.5 Hz, 3-H), 6.80 (1H, dd, J=9.1Hz, J=2.6 Hz, 10-H), 6.54 (1H, d, J=2.6 Hz, 8-H), 6.10 (1H, s, 6-H),5.12 (2H, s, CH₂ Ph), 4.03 (2H, t, J=6.4 Hz, ArOCH₂), 3.54 (2H, t, J=7.7Hz, HO₃ SCH₂ CH₂ CH₂ N), 3.51 (2H, q, J=7.1 Hz, NCH₂ CH₃), 2.91 (2H, t,J=7.2 Hz, HO₃ SCH₂), 2.44 (2H, t, J=7.3 Hz, CH₂ OBn), 2.14 (2H, m, HO₃CH₂ CH₂ CH₂ N), 1.80 (2H, m), 1.74 (2H, m), 1.52 (2H, m), 1.25 (3H, t,J=7.1 Hz, NCH₂ CH₃); δ_(C) (100.6 MHz; MeOH d₄) 184.97 (CO), 175.1 9(CO₂ Bn), 163.28, 153.85, 153.08, 148.11, 139.66, 137.73, 135.39,132.37, 129.58 (CH), 129.23 (CH), 128.40 (CH), 126.35, 119.20 (CH),111.98 (CH), 107.47 (CH), 97.48 (CH), 69.28 (CH₂), 67.23 (CH₂), 46.54(CH₂), 35.07 (CH₂), 26.65 (CH₂), 25.90 (CH₂), 25.87 (CH₂), 24.30 (CH₂),12.80 (CH₃); m/z (ES) 633 (MH⁺, 17%), 632 (M⁺, 43), 281 (30), 255 (49),177 (60), 130 (68), 100 (100); fluorescence in methanol strong.

6-(9-N-Ethyl-(3-sulfonylpropyl)amino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoicacid (XVII)

Ethyl6-(9(N-ethyl-(N-3-sulfonylpropyl))amino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoate(0.3 g, 0.55 mmol) was dissolved in ammonium dihydrogen phosphate (0.1M, 50 ml. pH 7) and porcine liver esterase (6.3 ml, 20000 units) added.The mixture was stirred at 37° C. for 14 days. The water was removedunder reduced pressure. The residue was dissolved in methanol and theenzyme extract filtered off through celite. The crude mixture waspurified by reverse phase chromatography on preadsorbed silica (methanolelution), to yield a dark red crystalline solid.

Yield (0.15 g, 53%); m.p. 222-225° C.; ν_(max) (KBr)/cm⁻¹ 3441, 3064,2934, 1732, 1640, 1619, 1594; λ_(max) (MeOH)/nm 557 (ε28648), 531(31100), 325 (6233), 261 (28726), 216 (21551); δ_(H) (250 MHz; MeOH d₄)7.74 (1H, d, J=8.8 Hz, 4-H), 7.49 (1H, d, J=2.3 Hz, 1-H), 7.22 (1H, d,J=9.1 Hz, 11-H), 6.82 (1H, dd, J=8.7 Hz, J=2.5 Hz, 3-H), 6.65 (1H, dd,J=9.1 Hz, J=2.3 Hz, 10-H), 6.31 (1H, d, J=2.3 Hz, 8-H), 5.86 (1H, S,6-H), 3.89 (2H, t, J=6.2 Hz, ArOCH₂), 3.50 (4H, m, HO₃ SCH₂ CH₂ CH₂ N,NCH₂ CH₃), 7.10 (2H, t, J=7.1 Hz, HO₃ SCH₂), 2.37 (2H, t, J=7.2 Hz, CH₂CO₂ H), 2.10 (2H, m, HO₃ SCH₂ CH₂ CH₂ N), 1.77 (4H, m), 1.50 (2H, m),1.22 (3H, t, J=7.0 Hz, NCH₂ CH₃); δ_(C) (100.6 MHz; MeOH d₄) 185.21(CO), 177.96 (CO₂ H), 163.71, 154.21, 153.57, 139.93, 135.77, 132.84(CH), 128.77 (CH), 126.89, 126.47, 119.65 (CH), 112.52 (CH), 107.79(CH), 105.32 (CH), 97.92 (CH), 69.78 (CH₂), 51.10 (CH₂), 49.07 (CH₂),47.03 (CH₂), 35.41 (CH₂), 30.49 (CH₂), 27.24 (CH₂), 26.39 (CH₂), 24.79(CH₂), 13.28 (NCH₂ CH₃); m/z (ES) 542 (M⁺, 22%), 255 (14), 177 (100);fluorescence in methanol strong.

EXAMPLE 7 Fluorescence Characterisation of Derivatised Molecules Effecton Fluoresence of Derivatisation and Linkage of Benzophenoxazines toBiological Molecules

Fluorescence spectra (uncorrected) were determined on 1 μM methanolsolutions of HPLC purified samples using a Hitachi F4500spectrofluorimeter. Relative fluorescence was determined from theemission curve at the peak excitation wavelength using the "area"function on the fluorimeter.

    ______________________________________                                                 Excitation peak                                                                            Emission peak                                                                            Relative peak                                   (nm) (nm) fluorescence                                                     ______________________________________                                        Nile Red 558          635        1                                              VII 558 633 0.70                                                              XI 558 633 0.72                                                               XVII 558 633 0.73                                                           ______________________________________                                    

The data clearly show that modification at the 2 and the 9 positionresulted in only a small decrease in fluorescence intensity compared tothe that of the parent compound.

Relative Fluorescence in Aqueous Environment of Nile Red and aDerivative Containing Water Solubilising Functions

Spectra were determined 1 hour after diluting 100 μM methanolicsolutions 100 fold into 50 mM Tris buffer pH 7.1. Relative fluorescencewas determined from the emission curve at the peak excitation wavelengthusing the "area" function on the fluorimeter.

The results showed a time dependent decrease in fluorescence of Nile Redin buffer. After 1 hour there was a 33 fold difference in Nile Redfluorescence intensity in methanol compared to that in water (seetable). This was associated with a large light scatter peak at theexcitation wavelength, presumably due to aggregation of the fluorophore.Compound XVII, which has a sulfonic acid group to provide watersolubilising properties, showed a fluorescence in water which wasreduced by only 3.6 fold compared to its fluorescence in methanol. Theintensity appeared not to vary with time.

    ______________________________________                                        Peak                    Peak                                                    excitation and Relative excitation and Relative                               emission fluorescence emission in fluorescence                                in methanol in buffer in                                                      (nm) methanol* (nm) buffer*                                                 ______________________________________                                        Nile Red                                                                             558/635    1.00      599/657  0.03                                       XVII 558/633 0.73 592/651 0.20                                              ______________________________________                                         *Relative to the fluorescence intensity of Nile Red in methanol.         

Normalised spectra of Nile Red and compound XI in methanol, and compoundXVII in buffer are shown in FIG. 3.

EXAMPLE 8 Synthesis of9-(N-2-carboxyethyl-N-ethyl)amino-5H-benzo[a]phenoxazin-5-one (XVIII)Synthesis of N-(2-methoxycarbonyl)ethyl-N-ethyl-m-aminophenol ##STR4##Step 1 Synthesis of N-ethyl-m-aminophenol from resorcinol

Resorcinol (200 g, 1.82 mol) was heated to 140-150° C. and stirred.Monoethylamine* was steadily passed through the melt for 32 hours. Theviscous oily product was stirred and washed with hot water (3×200 ml) toremove unreacted resorcinol.

The mono-ethylated product was purified by vacuum distillation (bp159-162° C. at 8 mm Hg).

GC-MS analysis confirms the product to be pure with very little startingmaterial and impurities present.

Yield=103.65 g, m/z (GC-MS) 137 (M⁺).

Step 2

Reaction with methyl acrylate

N-Ethyl-m-aminophenol (48 g, 0.035 mol), methyl acrylate (100 ml) andacetic acid (150 ml) were heated under reflux for 4 hours, after whichtime tic analysis (silica; dichloromethane-acetone 95:5) confirmed thecompletion of the reaction. The excess methyl acrylate/acetic acid wasremoved under vacuum at a temperature no greater than 70° C. Theresidual oil was then neutralised with NaOH solution, extracted with DCMand isolated.

Yield=45 g; m/z (GC-MS) 223 (M+), 208 (M⁺ -Me), 150 (M+--CH₂ CO₂ Me).

Synthesis of Dye (XVIII) Methyl Ester Derivative ##STR5##

N-Ethyl-m-aminophenol (4g, 0.0179 mol) was dissolved in water (6 ml) andconc. HCl (10 ml) and heated to 60° C., stirring vigorously. Thesolution was cooled to -5° C. and a solution of sodium nitrite (10 ml)added over 10 minutes (the solution was made up by mixing 5 ml of asaturated solution of NaCl in water with 5 ml containing 1.5 g sodiumnitrite (0.022 moles)). Another 5 ml of saturated NaCl solution wasadded and the solution stirred at 0° C. for 1 hour. The supernatantliquid was decanted off and the oily nitroso product used directly inthe next stage.

The nitroso derivative was dissolved in ethanol (40 ml) to which1-naphthol (3.0 g, 0.021 moles) was added. This solution was heatedunder reflux for 20 hours after which a distinct colour change fromyellow/brown to violet had occurred.

The dye methyl ester was isolated by column chromatography(silica/dichloromethane plus 5% acetone) and dried in a vacuumdessicator.

Yield=1.0 g, 15%; m/z (FAB) 377 (MH⁺), 399 (MNa⁺).

Hydrolysis to Give Dye (XVIII) ##STR6##

The methyl ester (0.5 g) was stirred in water (50 ml) and conc. HCl (15ml) at 80-90° C. for 3 hours.

The solution was neutralised to pH 5 with 30% NaOH and the solid productfiltered off and dried in a vacuum dessicator.

Yield=0.34 g; purity by tic satisfactory; UV λ_(max) (MeOH) 552 nm,λ_(emission) (562 nm excitation) (MeOH) 636 nm.; (10 mM HEPES pH 8.1)λ_(emission) (590 nm excitation) (10 mM HEPES pH 8.1) 661 nm; δ_(H) (300MHz; DMSO-d₆) 1.13 (3H, t, J7.0 Hz, Me), 2.57 (2H, t, J 7.0 Hz, CH₂ CO₂H), 3.51 (2H, br. q, CH₂ Me), 3.68 (2H, t, J 7.0 Hz, CH₂ CH₂), 6.29 (1H,s, 6-CH), 6.68 (1H, d, J 2.6 Hz, 8-CH), 6.83 (1H, dd, J 9.2 and 2.6 Hz,10-CH), 7.62 (1H, d, J 9.2 Hz, 11-CH), 7.71 and 7.81 (each 1H, m, 2 and3-CH), 8.11 and 8.54 (each 1H, d, J8.1 Hz, 1 and 4-CH) ppm m/z (MALDITOF) 362 (M⁺).

EXAMPLE 9 Labelling of Oligonucleotides with Dye (VII)

The DNA sequence T₆ was synthesised on an ABI 381A DNA synthesiser usingstandard reagents and cycles. The final monomer added to the 5' terminusof the resin bound oligomer was C6 aminolink (Glen Research) then thetrityl protecting group was removed under standard conditions. The dye(VII) (8.6 mg, 100 eq.) was dissolved in DCM (0.2 ml) thendiisopropylethylamine (3.5 μl, 100 eq.) was added followed by HBTU (7.5mg, 100 eq.). The solution was then added to the DNA synthesis column bydouble syringe methodology, i.e. two syringes attached to either side ofthe DNA synthesis column were used to repeatedly pass the reactionmixture through the column, for 2 h. Then the column was washed with DCM(2×20 ml). The resin bound DNA was then cleaved from the solid supportby treatment of the resin with K₂ CO₃ /MeOH (10% w/v) for 1 h., allvolatiles were then removed in vacuo, and the product was isolated byreverse phase HPLC (DYNAMAX C18) using the following eluant system (FIG.4).

Buffer A=0.1M Triethylammonium acetate

Buffer B=0.1M Triethylammonium acetate/MeCN (50/50 v/v)

    ______________________________________                                        Time/min.    Flow rate/ml/min.                                                                         % Buffer B                                           ______________________________________                                        0            4           0                                                      3 4 0                                                                         4 4 10                                                                        25 4 100                                                                      27 4 100                                                                      28 4 0                                                                      ______________________________________                                    

The isolated product was characterised by MALDI TOF MS m/z 2373.2 [C₉₂H₁₁₄ N₁₅ O₄₇ P₆ requires 2373.8] and UV λ_(max) (H₂ O) 260 and 600 nm.

Similarly the oligonucleotide primer5'-C6-amino-link-tgtaaaacgacggccagt-3' (SEQ. ID. NO.:1) was labelledusing the dye NHS ester (VIII) using a solution chemistry approach.

A mixture of the amino-linked primer (10 OD units) and dye NHS ester(VIII) (0.5 mg) in DMF (60 μl) and 0.2M carbonate buffer (55 μl) wasincubated for 16 hours at room temperature. The oligonucleotide was thenethanol precipitated twice and redissolved in 5% aqueous acetonitrile togive the required labelled oligonucleotide along with some unlabelledmaterial. UV λ_(max) 260 and 598 nm

EXAMPLE 10 Synthesis of a Range of Dye Labelled Peptide NH₂-AYVHDAPVRSLNK-OH (SEQ. ID. NO.:2)

The peptide is a substrate for Interleukin Converting Enzyme (ICE) whichcleaves the peptide between the DA residues.

The peptide was synthesised on a Applied Biosystems model 431A peptidesynthesiser using standard Fmoc chemistry. At the end of the synthesisthe N-terminal Fmoc group was removed, however the protected peptide wasleft attached to the solid support, in which form it was reacted withthe N-hydroxysuccinimidyl ester of dyes (I),(XVII) and (XVIII). Thelabelled peptide was then cleaved from the solid support using standardtechniques and then purified by reverse phase HPLC.

Synthesis of N-hydroxysuccinimidyl ester of dye (XVII)

Dye (XVII) (4 mg, 7.4 μg), N-hydroxysuccinimide (5 mg, 44 μg),N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimide methyl p-toluenesulphonate (9.3 mg, 22 μg), 4-dimethylaminopyridine (catalyticamount--one small crystal) were placed in a round bottomed flask towhich was added 1 ml of dry DMF. The mixture was stirred magneticallyovernight (18 hrs) with light excluded at 22° C.

Labelling of the peptide

32 mg of the ICE substrate peptide (equivalent to 8 μm peptide) wasweighed into a 1.5 ml screw top polypropylene V-vial to which was addedthe NHS reaction mixture followed by 20 μl of diisopropylethylamine. Thevial was placed on rollers with light excluded for 20 hrs at ambienttemperature (22° C.). The resin was then filtered off using a sinteredglass frit, washed with 5 ml dry DMF, 5 ml methanol and finally 5 mldichloromethane then dried in vacuo for 2 hrs.

Cleavage of the labelled peptide from solid support

The resin was placed in a small round bottomed flask to which was added2 ml of an ice-cold solution of trifluoroacetic acid (1.8 ml), water (50μl), ethanedithiol (50 μl) and thioanisole (100 μl). The mixture wasstirred magnetically for 90 minutes and allowed to warm to ambienttemperature. The mixture was then filtered through a glass wool plug andallowed to drip into 10 ml of ice cold diethyl ether. The dark blueprecipitate was spun down, the supernatant removed, the precipitateredissolved in 1 ml trifluoroacetic acid and reprecipitated in 10 ml icecold ether. The precipitate was spun down, washed twice with ether thendried in vacuo.

HPLC purification

The crude labelled peptide was dissolved in 1 ml water, filtered througha 0.45 μm Millipore filter and purified on a semi-prep. C-18 Vydaccolumn (code 218TP510) using a gradient of 0.1% TFA/water to 70% of 0.1%TFA/acetonitrile over 20 minutes and a flow of 4 ml/minute. Detectionwas at 230 nm.

Two major peaks were eluted, the first at 10.5 minutes was colourless,the second at 13.5 minutes was blue. This second peak was freeze driedto give 2.3 mg (1.15 μm) of a bright blue solid. Mass spectroscopy ofthis material gave a single peak at 1995 m.u. (calculated molecular wt.of dye (XVII) labelled peptide=1991.3)

The following dyes were also used to label the above manner usinganalogous procedures

Dye (II)

Dye (II) (3 mg) gave, after purification and freeze drying, 1 mg (0.6μm) of the labelled peptide as a bright blue solid. Mass spectroscopy ofthis material gave a single peak at 1820 m.u. (calculated molecular wt.of dye (II) labelled peptide 1812.7)

Dye (XVIII)

Dye (XVIII) gave, after purification and freeze drying, 6 mg (3.3 μm) ofthe labelled peptide as a bright blue solid. Mass spectroscopy of thismaterial gave a single peak at 1816 m.u. (calculated molecular wt. ofdye (XVIII) labelled peptide=1812.7)

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS:  2                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                    oligonucleotide primer                                                  <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: At position 1, N= C - #6-amino-link group             - - <400> SEQUENCE: 1                                                         - - ntgtaaaacg acggccagt             - #                  - #                      - # 19                                                                   - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic           peptide                                                                  - - <400> SEQUENCE: 2                                                         - - Ala Tyr Val His Asp Ala Pro Val Arg Ser Le - #u Asn Lys                    1               5 - #                 10                                   __________________________________________________________________________

What is claimed is:
 1. A benzophenoxazine compound having the formula##STR7## where X is O or NH or N-alkyl or N-aryl or N-alkenyl,Y is --NR¹R² or H, R¹ and R² are the same or different and each is C1-C12 aryl,alkenyl or alkyl or is -L-A, in at least one -L-A:L is a linker chain of0-20 carbon atoms and which may contain one or more O or N or S atoms,and A may be amine or amide or --CN or alcohol or thiol or carboxyl orsulfonate or phosphate, or a reactive group by means of which thebenzophenoxazine compound may be covalently linked to a biomolecule, ora group which enhances water solubility or provides electron donor orwithdrawal properties to modify the spectral characteristics of the dye,with the proviso that when L is zero, then A is not H, and each other-L-A is H or C1-C20 alkyl.
 2. A compound as claimed in claim 1 whereinthe benzophenoxazine ring 2-substituent L is --OC_(n) H_(2n) -- or--OCOC_(n) H_(2n) -- where n is 1 to
 20. 3. A compound as claimed inclaim 1, wherein in the benzophenoxazine ring 2-substituent L is --OC₅H₁₀ -- or --OCOC₄ H₈ --.
 4. A compound as claimed in claim 2, whereinthe -L-A- group at the benzophenoxazine ring 2 position is --OC_(n)H_(2n) COOH wherein n is 1 to
 20. 5. A compound as claimed in claim 2,wherein the -L-A- group at the benzophenoxazine ring 2 position is --OC₅H₁₀ COOH.
 6. A compound as claimed in claim 1, wherein at least onegroup R¹ or R² is --C_(n) H_(2n) SO₃ H where n is 1-12. 7.6-(9-N-Ethyl-(3-sulfonylpropyl)amino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoic acid.
 8. A compound as claimed in claim 1, wherein R¹ is--C_(n) H_(2n) COOH where n is 1-12. 9.9-(N-2-carboxyethyl-N-ethyl)amino-5H-benzo[a]phenoxazin-5-one.
 10. Acomplex of a biomolecule with a benzophenoxazine compound having theformula ##STR8## where X is O or NH or N-alkyl or N-aryl or N-alkenyl,Yis --NR¹ R² or H, R¹ and R² are the same or different and each is C1-C12aryl, alkenyl or alkyl or is -L-A, in at least one -L-A:L is a linkerchain of 0-20 carbon atoms and which may contain one or more O or N or Satoms, and A may be amine or amide or --CN or alcohol or thiol orcarboxyl or sulfonate or phosphate, or a reactive group by means ofwhich the benzophenoxazine compound may be covalently linked to abiomolecule, or a group which enhances water solubility or provideselectron donor or withdrawal properties to modify the spectralcharacteristics of the dye, with the proviso that when L is zero, then Ais not H, and each other -L-A is H or C1-C20 alkyl.
 11. A complex asclaimed in claim 1, wherein the biomolecule is a nucleoside ornucleotide or analogue thereof, or an aligonucleotide or nucleic acid.12. A complex as claimed in claim 10, wherein the biomolecule is anucleoside or nucleotide or analogue thereof, or an oligonucleotide ornucleic acid.
 13. A complex as claimed in claim 10, wherein thebiomolecule is a protein, peptide or amino acid.
 14. A complex asclaimed in claim 10, wherein the biomolecule is a polysaccharide,oligosaccharide or monosaccharide.
 15. A complex as claimed in claim 10,wherein the biomolecule is a drug or a small molecule.